PROJECT SUMMARY Recent advances in genome sequencing has enabled the detection of random somatic mutations, or neoantigens, in cancer cells from individual patient samples. Thus, neoantigens are ideal targets for cancer vaccination because they avoid harmful toxicities to healthy cells, and they are personalized based on patient samples. For successful vaccination, mRNA molecules need to be delivered to dendritic cells in the lymph nodes and spleen, which then present the antigen to T cells. This results in antigen-specific T cell activation that enables them to recognize cancer cells expressing the neoantigen of interest. However, the use of mRNA for vaccination is limited by molecular instability, in vivo delivery barriers, and insufficient T cell activation. The main goal of this proposal is to develop lipid nanoparticles (LNPs) to deliver neoantigen mRNA vaccines to DCs in vivo. LNPs improve upon mRNA delivery by protecting the molecules from degradation, enabling their cellular uptake, and providing tissue- and cell-specific accumulation. However, it is not well understood how LNP chemical composition and physical properties influence mRNA delivery to the lymph nodes, spleen, and to specific immune cell populations. Developing LNPs specifically for mRNA delivery is challenging because (i) only one LNP can be evaluated in a mouse at a time, and (ii) results evaluating cellular uptake of LNPs in vitro does not indicate in vivo results. This proposal will utilize high throughput molecular barcoding technology to screen a library of LNPs simultaneously in a single mouse. This will enable us to identify key structure:function relationships between LNP design and delivery, and it will provide LNPs that elicit strong immune responses for neoantigen vaccine delivery. Thus, I hypothesize that molecular barcoding will predict the LNP designs that yield the strongest antigen-specific T cell responses to treat cancer. In Aim 1, I will develop and evaluate molecular barcoding nanotechnology to assess LNP delivery to immune cells, and particular subtypes of dendritic cells, in the lymph nodes and spleens in vivo. In Aim 2, I will use the top-performing LNPs to deliver neoantigen vaccines and elicit the maturation of dendritic cells and activation of T cells to treat a mouse model of colon cancer. Moving forward, these highly modular platforms can be used to deliver multiple neoantigen mRNAs simultaneously, and they can be used as tools for studying the underlying immunobiology of specific populations of immune cells towards improving cancer vaccination.